DET1 regulates the proteasomal degradation of LHY, a component of the Arabidopsis circadian clock

Multiple photoreceptors contribute to the entrainment of the Arabidopsis circadian clock to daily cycles of light and darkness but little is known of the mechanisms by which these pathways affect the central oscillator. Here we investigate the epistatic interaction between DE-ETIOLATED 1 (DET1), a negative regulator of light-regulated gene expression, and LATE ELONGATED HYPOCOTYL (LHY), one of the core components of the circadian oscillator. The daily onset of LHY gene expression was advanced by approximately 4 h in det1-1 mutant plants, suggesting that the wild-type DET1 protein might function to repress its transcription during the subjective night. lhy-1 det1-1 double mutants exhibited arrhythmic expression of the CAB gene in constant light, similar to the lhy-1 mutant parent. However, additive effects of the lhy-1 and det1-1 mutations on CAB2 expression patterns were revealed under diurnal light-dark cycles. Since the lhy-1 mutation causes aberrant, constitutive transcription of LHY from a constitutive viral promoter, this observation indicated that effects of DET1 were not mediated through the regulation of LHY transcription. Furthermore, the light-driven, rhythmic accumulation of the LHY protein in the lhy-1 mutant was altered by the det1-1 mutation, suggesting that DET1 might regulate LHY expression at the post-transcriptional level. In vitro protein degradation assays demonstrated that the LHY protein is turned over rapidly through the proteasome pathway. Similar degradation was observed whether plant tissue was harvested during the light or dark portion of the diurnal cycle, but the process was significantly accelerated in det1-1 mutant extracts. These results indicate that the wild-type DET1 protein acts to inhibit the proteolytic turnover of the LHY protein, and suggest a mechanism for the period-shortening effect of the det1-1 mutation. These findings add to recent evidence suggesting a role for DET1 in a ubiquitination pathway and identify a substrate for DET1-regulated protein turn-over